Sterols in general, mainly plant sterols or phytosterols, currently have numerous applications and are most commonly used as food ingredients. Such applications require the use of phytosterols dispersed in aqueous media such as dairy beverages, aqueous fancy drinks, fruit juices, or in lipidic media like margarines and mayonnaise among others. Since the applications of dispersed phytosterols are so numerous, it is important to have highly stable aqueous dispersions that can be stored for long periods before being used in diverse applications and simultaneously provide homogeneous products when used in specific applications, wherein phytosterols remain dispersed, no phase separation takes place and the products have good palatability. The interest of the food industry for the elaboration of phytosterol-containing products is a result of the proven capacity of phytosterols to inhibit the absorption of dietary cholesterol.
Since the 1930's, it has been known that plasmatic cholesterol levels are affected by the ingestion of phytosterols. Later research conducted around the 1950's has definitively shown, both in animals and humans, that phytosterols are very efficient at inhibiting the absorption of externally provided cholesterol, i.e. dietary cholesterol. Consequently, people ingesting phytosterols with their cholesterol-containing food have a lower level of serum cholesterol than those whose food does not contain phytosterols. As a result, there has lately been a great interest on the elaboration of phytosterols-containing food products as an effective and low-cost way to provide phytosterols for those in need thereof.
Although phytosterols are not soluble in aqueous media and are hardly soluble in fatty media, phytosterols have not to be soluble in the ingested food to exert their hypocholesterolemic effect, but are capable of exhibiting their cholesterol-lowering effect when dispersed in these media. Although the cholesterol-lowering effect of phytosterols is well known in the state of the art, it has been surprisingly found that phytosterol dispersions prepared according to the process of the present invention have a significantly higher cholesterol-lowering effect than similar dispersions prepared according to processes known in the state of the art and have higher cholesterol-lowering effects achieved using one of the most known commercial products having this effect, namely phytostanol esters.
Usually, in the preparation of aqueous dispersions of phytosterols, one or more surface active agents are used as emulsifiers which are added to the preparation in a proportion over 20% by weight in relation to phytosterols, which is typically over 50%. Most of the water-dispersible phytosterol powders contain about 50% phytosterols that consists of one part phytosterols and one part emulsifiers or other additives. Although dispersible phytosterol powders containing up to 85% by weight of phytosterols can be found in the market, said powders have a very poor dispersibility in water, and once suspended in water at 1% by weight they separate from the aqueous phase easily and settle down in less than one hour. In order to stabilize such dispersions, they require higher amounts of emulsifiers and further processing, usually by high-pressure homogenization, which implies additional more complicated formulation steps. Currently, there are no commercially available stable aqueous dispersions of phytosterols that could be used as functional food ingredients, mainly because they settle down easily, have a low phytosterol content in the dispersion (high water content) and have a limited shelf life (less than one month). Therefore, dispersions are usually dehydrated and commercialized as resuspendable powders. In the following sections, the terms surface-active agent and surfactant are used interchangeably.
Food emulsifiers constitute only a relatively small group of the emulsifier family, and many of them have dietary restrictions concerning their daily intake, which in some cases such as polysorbates can be as low as 25 mg/kg. This limits the combination of emulsifiers that can be used in the phytosterol dispersion processes. This problem is more serious when more than one surfactant must be used in the dispersion, when they have to be incorporated in food or pharmaceutical products.
The suggested daily intake of phytosterols as cholesterol-lowering agents is between 0.8 and 2.0 g, therefore, in case that they are accompanied by high levels of food emulsifiers, the total daily intake of food emulsifiers considering emulsifiers present in other food items as well might very well surpass the maximum allowable daily intake for some emulsifiers or in the case of mono- or diglycerides they might significantly increase the overall daily calorie intake.
There is a need, therefore, to develop solutions to obtain highly stable aqueous phytosterol dispersions with the lowest possible emulsifier/phytosterol ratio, using the whole range of food emulsifiers, including predominantly hydrophobic and predominantly hydrophilic surfactants or their mixtures.
There are a large number of processes disclosed in the prior art aimed at the preparation of aqueous phytosterol suspensions or dispersions and their use as an ingredient of hypocholesterolemic food. Some of the most relevant prior art documents of the past 20 years concerning the subject matter disclosed in the present application are shown in Table 1.
In the present invention the term emulsion has the usual meaning, that is, an emulsion is a mixture of two immiscible liquids wherein one of the liquids, known as the dispersed phase, is dispersed within the other liquid, known as the continuous phase. Therefore, an aqueous phytosterol emulsion means a dispersion of liquid phytosterols in an aqueous medium, while the term aqueous dispersion of phytosterols refers to the dispersion of solid phytosterols in an aqueous medium. To form a stable emulsion, the emulsion should contain one or more surface-active agents and sometimes also other components known generically as protective colloids. Surface-active agents or surfactants are substances that have both hydrophilic and hydrophobic portions in the same molecule, i.e. they have an amphiphilic character. This means that they have the tendency to concentrate in the interface, so reducing the interfacial tension. To characterize the degree of amphiphilicity of an amphiphilic molecule, an empirical numerical scale ranging from 1 to 40, denominated the HLB value, is used. The lower the HLB value, the more hydrophobic the molecule is and, conversely, the higher the HLB value, the higher is the hydrophilic character of the molecule. There are a large number of surface-active agents, both natural and synthetic, which are useful for a variety of applications. An emulsifying agent is a surface-active agent utilized in the preparation of emulsions with the aim of stabilizing the emulsion. It has been found empirically that those surface-active-agents having HLB number ranging from 8 to 18 are useful for stabilizing oil in water emulsions (o/w) wherein the disperse phase is oil and the continuous phase is water or an aqueous medium. On the other hand, surface-active agents which have an HLB number ranging from 3 to 6 are useful for stabilizing water in oil emulsions (w/o), wherein the disperse phase is water and the continuous phase is oil. In the field of emulsions, the term oil means any liquid that is immiscible with water. The term aqueous medium means either water or a solution, dispersion or w/o emulsion containing at least 30% of water by weight of solution, dispersion or emulsion.
TABLE 1State of the art processes for the preparation of phytosterol dispersions.Document numberInventorTitleEP1645267A2Behnam, DariushMethod for producing an active ingredient concentrate andactive ingredient concentrate.20070031570Binder, Thomas P.Hydrothermically processed compositions containingphytosterols.6623780Stevens, Luke AlanAqueous dispersible sterol products.20070141224Zawistowski, JerzyCompositions comprising one to more phytosterols and/orphytostanols, or derivatives thereof, and high HLBemulsifiers.20060035009Gaonkar, AnilkumarCompositions and processes for water-dispersible phytosterolsand phytostanols.WO/2002/065859Auriou, NicolasStabilized dispersions of phytosterol in oil.20030165572Auriou, NicolasWater-dispersible encapsulated sterols.20050170064Yoon, Won-taePlant sterol-containing food, and method for preparing thesame.WO/2003/077680Yoon, Won-taeMixing powder of plant sterol and emulsifier, and method forpreparing the same.20070231447Fleckenstein, MichaelSterol compositions and methods of making the same.EP1575378A1Auweter, HelmutPulverulent phytosterol formulations.6316030Kropf, ChristianUse of nanoscale sterols and sterol esters.20040033202Cooper, Eugene R.Nanoparticulate sterol formulations and novel sterolcombinations.WO/1999/063841Stewart, David JohnCompositions comprising phytosterol and 7 or phytostanolhaving enhanced solubility and dispersibility.20050244488Spilburg, Curtis A.Methods and formulations for enhancing the absorption andgastrointestinal bioavailability of hydrophobic drugs.6110502Burruano, BridMethod for producing water-dispersible sterol formulations.WO/2002/017892Auriou, NicolasStabilized dispersions of phytosterol in oil.7306819Lerchenfeld, ErichBeverages containing plant sterols.7335389Lerchenfeld, ErichBeverages containing plant sterols.20070014819Wu, Wen-tengMethod of emulsifying phytosterol by natural saponin,emulsion prepared thereby and water-dispersible phytosterolpowder product.20040142087Lerchenfeld, Erich PBeverages containing plant sterols.WO/2003/103633Cooper, Eugene RNanoparticulate sterol formulations and sterol combinations.WO/2003/094891Spilburg, Curtis A.Methods and formulations for enhancing the absorption andgastro-intestinal bioavailability of hydrophobic drugs.20020064548Yoon, Won-taeMethods for dispersing plant sterol in aqueous phase and plantsterol-dispersed beverages.WO/2003/000075Dyer, MatthewMethod for manufacturing of free-flowing powder containingwater-dispersible sterols.WO/2001/053320Stewart, David JohnCrystalline composites comprising phytosterols andphytostanols or derivatives thereof.WO/2007/124597Stewart, David JohnCompositions comprising one or more esterified phytosterolsand/or phytostanols into which are solubilized on or moreunesterified phytosterols and/or phytostanols, in order toachieve therapeutic and formulation benefits6113972Corliss, GlennPhytosterol-protein complexes.WO/2006/074752Veldhuizen, YvonneSachets comprising plant sterol.20030003131Dyer, MatthewMethod for manufacture of free-flowing powder containingwater-dispersible sterols.
There are numerous disclosures in the state of the art concerning the preparation of dispersible phytosterols in aqueous media comprising mechanical dispersion of phytosterols in aqueous media in the presence of one or more surface-active agents, the dissolution of phytosterols in some suitable solvent, either sub- or super-critical followed by the dispersion of the dissolution in an aqueous phase, the preparation of water-soluble phytosterol complexes, the thermal dissolution of phytosterols in one or more surface-active agents followed by the dispersion of the dissolution in an aqueous media, the direct mixing of phytosterols with an aqueous solution of some surface-active agent followed by the injection of live steam at high temperature and many others.
With the exception of the process disclosed in the US Patent Appl. 20060035009 in which phytosterols are solubilized by preparing a water-soluble phytosterol-carbohydrate complex, the general approach for obtaining phytosterol preparations has been either by preparing water-dispersible phytosterol powders or aqueous phytosterol dispersions to get hypocholesterolemic effects. Ostlund, in the U.S. Pat. Nos. 5,932,562 and 6,063,776, also discloses processes for obtaining water-soluble phytosterols, but as shown in the Example 4 of the '562 patent, powdered sitostanol preparations did not exhibit cholesterol-lowering effects in human subjects. With the exception of the three documents above, the state of the art discloses a variety of methods either for the preparation of water-dispersible phytosterol powders or aqueous phytosterol dispersions, which in general are more readily obtained than water-soluble phytosterols. But as shown by Ostlund in the '562 patent, the efficacy of phytosterol powders or dispersions as cholesterol-lowering agents seems to depend on their preparation process.
When comparing the cholesterol-lowering effect of aqueous dispersions of phytosterols or water-dispersible phytosterol powders prepared according to the present invention with those prepared according to processes known in the state of the art, a significantly higher cholesterol-lowering effect has been observed in the first case, which would confirm that the cholesterol-lowering effect of solid phytosterols ingested as water-dispersible powders or aqueous phytosterol dispersions seem to depend, though unpredictably, on the process of their preparation.
There are numerous well-known methods for the dispersion of immiscible liquid phases to prepare emulsions. The preferred methods utilized in the present invention are those that resort to rotor/stator homogenizers such as colloid mills, stirred vessels and pressure homogenizers. This equipment is capable of providing high shear stress and shear rate allowing the preparation of aqueous phytosterol emulsions that give rise to aqueous phytosterol suspensions upon cooling with characteristics that are surprisingly different from the dispersions prepared using any of the processes of the state of the art.
The processes of the state of the art do not disclose any phytosterol dispersion process wherein phytosterols, an aqueous medium and one or more food-grade surface-active agents, with a low surface-active agent to phytosterols ratio and having significant phytosterol concentrations for use in the food or pharmaceutical industry, are dispersed at temperatures above the melting point of phytosterols to form an emulsion, followed by cooling down said emulsion to originate a phytosterol dispersion in aqueous medium.
On the contrary, this procedure has been discouraged in the documents of the state of the art considering the high melting point of phytosterols. In fact, in the U.S. Pat. No. 6,623,780 (Sep. 23, 2003) entitled “Aqueous dispersible sterol product”, the inventors (Stevens and Schmeltzer) explicitly state (see column 3, line 10 and following) that “The primary difficulty in formulating sterols is their high melting point. Commercially available food-grade sterols typically have a melting point range of 120° C.-140° C. This makes their dispersion in water very difficult because the sterols cannot be dispersed in water as liquid sterols without resorting to high temperatures and associated high pressures.”
In order to overcome what they considered a difficulty, the inventors resorted to mixing sterols, monoglycerides and a polysorbate such as Tween 60. This gives rise to a mixture with a lower melting point, which is around 75° C. The mixture is then cooled down, preferably by spraying the mixture into an air stream. The resulting sterol-emulsifier powder can subsequently be dispersed in an aqueous medium and be subjected to high-shear homogenization to form an emulsion, i.e. the incorporation of sterols into food matrixes requires the use of subsequent homogenization steps. According to the type and proportions of the claimed surface-active agents, the HLB value of the surface-active agent mixture ranges from 4 to 6 and the claimed ratio of emulsifiers to sterols ranges from 111% to 170%. The inventors have found that a dispersion prepared according to their invention, containing sterols, monoglycerides and polysorbates, is stable in pure water for several weeks.
By contrast, aqueous phytosterol dispersions prepared in accordance with the processes of the present invention have a shelf life of more than one year without any noticeable phase separation or settling during said period and are easily incorporated into liquid food items such as dairy beverages, aqueous fancy drinks, fruit juices and the like.
One of the disadvantages of the process disclosed in U.S. Pat. No. '780 is that in order to prepare a more or less stable aqueous phytosterol dispersion several steps are required i.e. mixing the sterols with one or more emulsifying agent, melting the mixture formed, spray-drying the melted product, and dispersing the resulting powder in an aqueous medium under high shear at a temperature higher than the melting point of the powder. Besides, in order to get a significant melting point reduction of the mixture of sterols and emulsifiers, a similar or higher amount of emulsifiers is required relative to the sterols utilized. In addition, a shelf life of a few weeks could be insufficient for certain applications. Furthermore, the range of usable emulsifiers is rather restricted in the disclosed process, leaving numerous important food emulsifiers out of the application field. Additionally, the large excesses of emulsifiers that accompany the sterols might contribute significantly to the calorie content of the food articles in which they are incorporated, an undesirable feature for cholesterol-lowering foods.
Yoon et al. in the US Patent Applications 20040029844, 20040170064 and 20020064548 disclose a similar process for the preparation of aqueous phytosterols, resorting to lower ratios of emulsifier/phytosterol that those used by Stevens and Schmeltzer in U.S. Pat. No. '780 but at the expense of using higher temperatures, closer to the melting point of sterols, which was precisely the problem that the inventors of the U.S. Pat. No. 6,623,780 wanted to avoid.
In the above quoted patent applications, the disclosed processes consist in mixing sterols with several emulsifiers at temperatures between 130° C. and 140° C. as illustrated in the Examples 1, 2 and 3 of the Patent Application 20040029844, in order to form a melted mixture. This mixture can be dispersed in an aqueous medium or drink, preferably between 70° C. and 90° C. The process is carried out in a mixer-stirrer with a rotation speed of 6,800 rpm to 10,000 rpm. The inventors state that, “after the stirring process, a homogenizing process is needed to pulverize aggregated micelles”.
The emulsifiers used were sucrose stearyl ester, sorbitan lauryl ester, sodium stearyl lactylate, polyglycerine monostearate and monoglyceryl citrate, in amounts varying from 17% to 85%, relative to sterols. The range of HLB values of these emulsifiers ranges from 8 to 15.
As shown in the Comparative Examples 1, 2 and 3 of the Patent Application 20040029844, when the emulsifiers used were sodium stearyl lactylate, polysorbate or monoglyceryl citrate, the resulting dispersions were unstable. According to the Examples, only sucrose stearyl ester (HLB 11) and lauryl sorbitan (HLB 8.6) and their mixtures were effective stabilizers. Therefore, the range of emulsifiers suitable for carrying out the disclosed invention is rather limited. According to the Examples of the present invention, the sterol dispersion prepared according to the Example 1 of the Application 20040029844 without the homogenization step (assay 5) is unstable, and when the homogenization step is included (assay 6), the resulting dispersion has considerably less stability than the sterol dispersion prepared according to the present invention. In addition, the process disclosed in the Application 20040029844 does not allow for the preparation of aqueous phytosterol dispersions with high solid contents.
Binder, in the US Patent Application 20070031570, discloses a method for the preparation of aqueous phytosterol dispersions by mixing water, phytosterols and an emulsifier and then heating the mixture with live steam between 100° C. and 200° C. for a period ranging from 2 seconds to 10 minutes, preferably from 30 seconds to 3 minutes. The heating process exerts a characteristically low shear stress upon the mixture. Then, the mixture is either cooled down in flash cooler or is further homogenized at a high pressure ranging from 2000 psi to 8000 psi, preferably twice. The specification does not disclose the homogenization temperature, but in the Example 1, after a hydrothermal process at 152° C., the mixture is cooled down to 79° C. and subjected to a two-stage homogenization step. Hence, the real homogenization is carried out on an aqueous dispersion of solid sterols.
To carry out the disclosed process, food-grade emulsifiers with low HLB values (lower than 5), such as lecithins, distilled mono and diglycerides and the like, are used. The amount of emulsifiers relative to the phytosterols in the Examples 1 and 2 was 199%.
Among the disadvantages of the disclosed process the following can be set forth: the range of emulsifiers utilizable is rather limited to emulsifiers with HLB values less than 5, which leaves out important emulsifiers required in certain applications; the product before the homogenization step is very unstable, and even after this homogenization step the product still has low stability; in addition, the product uses large excesses of emulsifiers in relation to the amount of phytosterols, which might be disadvantageous for its application in cholesterol-lowering foods which are usually required to be low-calorie foods as well.
Another disadvantage of the disclosed process, common to other processes in the state of the art, is inherent to the homogenizing step of aqueous dispersion of solid sterols due to the serious abrasion caused to the homogenizer valves. In the process of the present invention, homogenization is carried out over sterol emulsions, not dispersions. The consequence of this is not only the expected lack of abrasion, but also, unexpectedly, the resulting dispersion turns out to have surprising characteristics, such as a higher stability, the possibility of using a large range of emulsifiers at very low concentrations relative to the phytosterols and in the presence of large phytosterol concentrations.
It is evident that there is no process in the state of the art to prepare aqueous phytosterol suspensions having a very high stability, stabilized by one or more surface-active agents selected from a wide array of emulsifiers comprising both hydrophilic and hydrophobic emulsifiers with HLB values ranging from about 1 to about 20, that allows the production of dispersions with a low phytosterol content and also dispersions with more than 10%, preferably more than 20% phytosterols based on the weight of the aqueous dispersion.
The process disclosed in the present invention allows the preparation of highly stable phytosterol dispersions using a wide array of food-grade emulsifiers and their mixtures, with HLB numbers between 1 and 20, with low emulsifier/phytosterol ratio, and is an extremely simple process that can be satisfactorily carried out in a single step. The concentration range of phytosterols in the dispersions of the present invention ranges from 0.1 g to 400 g of phytosterols dispersed per liter of dispersion, which are two orders of magnitude higher than the highest concentrations achieved for any stable phytosterol dispersion disclosed in the state of the art. This is another advantage of the process of the invention, since aqueous dispersions with high phytosterol content are preferable in certain applications wherein high concentration of dispersed phytosterols are required, such as e.g. in margarine additives. Likewise, when the aim is obtaining a dispersible powder, it is preferable to begin with a dispersion that has the lowest water content possible in order to reduce the difficulties related to water removal.
Although the present invention was motivated by the detected necessity of providing a process to obtain phytosterol dispersions wherein the product of the process could overcome all the disadvantages of the phytosterol dispersions obtained using the processes disclosed in the state of the art, it has been found that dispersions prepared according to the process herein disclosed, both aqueous dispersions as well as resuspended powdered phytosterols, unexpectedly and surprisingly exhibited a significantly higher cholesterol-lowering effect with respect to the effect exhibited by dispersions of the state of the art, as will be shown in Example 16.